Soil bacteria of the genera Rhizobium and Bradyrhizobium, members of the family Rhizobiaceae, are capable of infecting plants and inducing a highly differentiated structure, the root nodule, within which atmospheric nitrogen is reduced to ammonia by the bacteria. The host plant is most often of the family Leguminosa. Bradyrhizobium, also sometimes designated "slow-growing" rhizobia (Jordan, D.C. (1982) Int. J. Syst. Bacteriol. 32:136), includes the commercially important soybean nodulating strains of Bradyrhizobium japonicum, the symbiotically promiscuous rhizobia of the "cowpea group," and some strains that can nodulate non-legumes, such as Bradyrhizobium sp. Parasponia. Rhizobium species, also sometimes designated "fast-growing" rhizobia, include among others R. trifolii (plant host: clover), R. meliloti (plant host: alfalfa), R. leguminosarum (plant host: pea). Rhizobium strains generally display a narrow host range.
Within the species Bradyrhizobium japonicum, a number of distinct serogroups including, for example, those designated as USDA 123, USDA 110 and USDA 138, are recognized. Strains belonging to the different serogroups have been found to display quantitatively different symbiotic or nodulation properties (Keyser and Griffen (1987) Beltsville Rhizobium Culture Collection Catalogue, U.S. Department of Agriculture). For example, B. japonicum USDA 110 are more effective for nitrogen fixation than strains of USDA 123, but USDA 123 strains appear to be more competitive for infection and nodule occupancy than USDA 110 strains.
The process of plant-host recognition, infection and effective nodule formation is complex and involves the coordinated expression of a number of genes in the bacterial symbiont and the host plant. The genetics of the symbiosis and nitrogen-fixation have been the focus of extensive research in recent years in both Rhizobium and Bradyrhizobium strains. Bacterial genes encoding nitrogenase (nif genes), affecting nitrogen fixation in general (fix) and nodule development and, at least in part, host range (nod) have been identified and in many cases located and cloned (see for example recent reviews: Broughton, W. J. (ed.) (1982) Nitrogen Fixation, Volumes 2 and 3 (Clarendon Press, Oxford); Puhler, A. (ed.) (1983) Molecular Genetics of the Bacteria-Plant Interaction (Springer-Verlag, Berlin); Szalay, A. A. and Legocki, R. P. (eds.) (1985) Advances in Molecular Genetics of the Bacteria Plant Interaction (Cornell University Publishers, Ithaca, New York); Long, S. R. (1984) in Plant Microbe Interactions, Vol. 1, Kosuge, T. and Nester, E. W. (eds.) (Macmillan, New York) pp. 265-306; Verma, D. P. S. and Long, S. L. (1983) International Review of Cytology (Suppl. 14) Leon, K. W. (ed.) (Academic Press, N.Y.) pp. 211-245).
The nodulation genes and their regulation have been of particular interest in efforts to understand the mechanism of selective infection of particular host plants. A set of genes (nod) associated with a nodulation defective phenotype (NOd.sup.+) has been identified on the Sym (symbiotic) plasmids of strains of Rhizobium. The "common" nod genes, designated nodA, B and C, which are associated with the early stages of infection and nodulation, are structurally conserved among Rhizobium strains. In R. meliloti, R. leguminosarum, and R. trifolii, the nodA, B and C genes are organized in a similar manner and are believed to be coordinately transcribed as a single genetic operon. The DNA region adjacent and 5' to nodA has been found to contain a fourth nodulation gene, designated nodD, which is transcribed divergently from the nodABC operon (Egelhoff et al. (1985) DNA 4:241-248; Jacobs et al. (1985) J. Bacteriol. 162:469-476; Rossen et al. (1984) Nucl. Acids Res. 12:9509-9524). NodD has been found to function in the regulation of expression of nodABC and other nodulation genes (Mulligan and Long (1985) Proc. Natl. Acad. Sci. USA 82:6609-6613; Rossen et al. (1985) EMBO J. 4:3369-3373; Innes et al. (1985) Mol. Gen. Genet. 201:426-432). Comparisons of the DNA sequences and the deduced amino acid sequences of the encoded nodD product confirm the presence of significant sequence conservation of these genes among strains of Rhizobium. NodD mutants in the various species of Rhizobium do not, however, display the same nodulation phenotypes (some are Nod.sup.-, and others "leaky" nod.sup.- or delayed in nodulation) (Downie et al. (1985) Mol. Gen. Genet. 198:255-262; Schofield et al. (1983) Mol. Gen. Genet. 192:459-465; Jacobs et al. (1985) J. Bacteriol. 162:469-476; Gottfert et al. (1986) J. Mol. Biol. 191:411-420). It now appears that many species of Rhizobium carry multiple nodD-like genes, on their Sym plasmids (Rodriquez-Quinones et al. (1987) Plant Mol. Biol. 8:61-75; Appelbaum et al. (1985) in Nitrogen Fixation Research Progress (Martinus Nijhoff Publishers, Dordrecht, The Netherlands) pp. 101-107; Gottfert et al. (1986) J. Mol. Biol. 191:411-420; Appelbaum et al. (1988) J. Bacteriol. 170:12-20). Another similarity in the nod region(s) of Rhizobium strains is the presence of conserved sequence elements within the promoter regions of certain inducible nod genes. These conserved sequences, first identified in the nodABC promoter region, are termed the nod-box and are believed to function in induced nod gene expression, possibly as regulatory protein binding sites (Scott et al. (1985) in Nitrogen Fixation Research Progress, Evans et al. (eds.), Martinus Nijhoff Publishers, Dordrecht, The Netherlands, p. 130; Rolfe et al. ibid, pp. 79-85; Kondorosi et al. ibid, pp. 73-78).
No Sym plasmids have been associated with Bradyrhizobium strains. The nitrogenase and nodulation genes of these bacteria are encoded on the chromosome. Bradyrhizobium strains contain nodulation genes which are reported to functionally complement mutations in Rhizobium and which show significant structural homology to nodulation gene regions of R. meliloti and R. leguminosarum (Marvel et al. (1985) Proc. Natl. Acad. Sci. USA 82:5841-5845; Russel et al. (1985) J. Bacteriol. 164:1301-1308). In Bradyrhizobium japonicum strains USDA 110 and 123, nodABC and D gene structural homologs have been identified which are organized in a manner similar to their homologs in Rhizobium strains (Appelbaum, U.S. patent application Ser. No. 875,297, filed Jun. 17, 1986). NodD is read divergently from nodAB and C which are organized in a single operon. The untranscribed region between nodD and nodA also contains a copy of the conserved nod-box (Appelbaum, U.S. patent application serial number 875,297; Stacy et al. (1987) in Molecular Genetics of Plant-Microbe Interactions; Verma and Brisson, (eds.) (Martinus Nijhoff Publishers, Dordrecht, The Netherlands) pp. 197-201). In contrast to Rhizobium strains, Bradyrhizobium japonicum strains contain another open reading frame, designated ORF, between nodD and nodA which is believed to be part of the nodABC operon (Appelbaum, U.S. patent application serial number 875,297). A second nodD homolog, nodD2, has been identified in Bradyrhizobium japonicum strains USDA 110 and 123, which is positioned about 640 bp downstream from and is transcribed in the same direction as nodD1 (Appelbaum, U.S. patent application Ser. No. 875,297). Both nodD genes may be coordinately transcribed. B. japonicum having a mutation in nodD1 only displays a delayed nodulation phenotype, while those having a mutation in nodD2 only show an apparent wild-type nodulation phenotype (see also Nieuwkoop et al. (1987) J. Bacteriol. 169:2631). However, mutants carrying a double mutation in both nodD genes display a more pronounced delay than is observed in nodD1 single mutants. The NodD double mutants do, however, remain nod.sup.+ (Appelbaum, U.S. patent application Ser. No. 875,297). Other nodD homologs may be present in the B. japonicum genome.
It has long been suggested that an exchange of signals between plants and bacteria is requisite for mutual recognition and coordination of the steps of infection and nodulation (Nutman, P. S. (1965) in Ecology of Soil Borne Pathogens, eds. F. K. Baker and W. C. Snyder, University of California Press, Berkeley, p. 231-247; Bauer, W. D. (1981) Ann. Rev. Plant Phys. 32:407-449; Schmidt, E. E. (1979) Ann. Rev. Microbiol. 33:355-376). It is now known, for example, that in both Bradyrhizobium and Rhizobium chemical factors contained in legume exudates induce the expression of nodulation genes.
In Rhizobium the expression of nodABC, node and F, and other nodulation genes is reported to be induced in the presence of legume exudates (Mulligan and Long (1985) Proc. Natl. Acad. Sci. USA 82:6609-6613; Rossen et al. (1986) EMBO J. 4:3369-3373; Innes et al. (1985) Mol. Gen. Genet. 201:426-432). Only very low levels of expression of these genes are reported in the absence of exudate. In contrast, nodD expression is reported to be constitutive and not inducible by such exudates. The expression of nodD is reported to be necessary in addition to exudate factors for the expression of the nodABCEFGHI and J genes. In some cases, nodD is reported to regulate its own expression (autoregulation).
The specific components of legume exudate that act to induce nodulation genes in several species of Rhizobium have been identified as flavonoids. Luteolin was reported to be the component of alfalfa exudates that induces nod ABC expression in R. meliloti (Peters et al. (1986) Science 233:977-980) Three clover exudate constituents: 4',7-dihydroxyflavone, geraldone and 4'-hydroxy-7-methoxyflavone were reported to induce the nodulation genes of R. trifolii (Redmond et al. (1986) Nature 323:632-635). Two pea exudate components: eriodictyol, and apigenin-7-O-glucoside were reported to induce the nodulation genes of R. leguminosarum (Firmin et al. (1986) Nature 324:90-92; Zaat et al. (1987) J. Bacteriol. 169:198-209). In addition, molecules having structures related to those of the inducer found in exudate were assessed for their ability to induce. Inducers of Rhizobium nodulation genes appear in general to be limited to certain substituted flavonoids, and the range of compounds to which a Rhizobium responds is species specific. Since host range is used to classify Rhizobium strains into different species, this suggests that differential response to inducer molecules is involved in the mechanism of determination of host range.
Two isoflavone components of soybean exudate, daidzein and genistein, have been reported to be inducers of the nodulation genes of B. japonicum strains 110 and 123 (Kosslak et al. (1987) Proc. Natl. Acad. Sci. USA 84:7428-7432. Several other isoflavones were found to be inducers (7-hydroxyisoflavone, 5,7-dihydroxyisoflavone and biochanin A) or weak inducers (formononetin and prunetin) of the B. japonicum nod genes In addition, two flavones: 4',7-dihydroxyflavone and apigenin which induce certain Rhizobium nod genes were also found to induce the B. japonicum nod genes. It is interesting to note that isoflavones were reported to be antagonists of induction of nodulation genes of Rhizobium strains (Firmin et al. (1986) Nature 324:90-92).
It has been reported that nodulation host specificity is at least in part mediated by a selective interaction of host plant factors with the specific nodD gene(s) of a particular rhizobium to stimulate nod gene expression and effect nodulation (Spaink et al. (1987) Nature 328:337-340; Hong et al. (1987) Nucl. Acids Res. 15:9677-9690). While the nodD genes of various rhizobia have significant homology, they are much more conserved in the amino-terminal portion than in the carboxy-terminal portion of the coding region. Any functional differences between nod gene products is expected to reside in the carboxy-terminal portion of the protein. Horvath et al. (1987) EMBO J. 6:841-848 have reported the construction of a chimeric nodD gene having the 5'-end of the nodD1 of a broad host range Rhizobium which nodulates the tropical legume siratro (MPIK3030) and the 3'-end of the nodD1 of R. meliloti. When this chimeric gene was introduced into a nodD.sup.- double mutant (both nodD genes inactivated) of R. meliloti, the transconjugant nodulated alfalfa normally. When this chimeric gene was introduced into a nodD' mutant of MPIK3030, the transconjugant strain did not nodulate siratro.
Burn et al. (1987) Genes & Development 1:456-464 also link nodD to the inductive response to inducer as well as antagonist (or anti-inducer) molecules. They report nodD mutants of Rhizobium leguminosarum that are affected in their ability to activate nod gene expression in response to inducer molecules. One class of mutants described, designated class IV, are reported to display nod gene induction in the absence of inducer molecules and enhanced levels of nod gene induction in the presence of inducer; what is herein designated as a hyperinducible phenotype. In addition, this class of mutants also displayed altered response to molecules which were antagonists of induction in the wild-type parent strain. However, class IV nodD mutants were reduced in nodule number compared to the wild-type and, furthermore, they did not fix nitrogen (fix.sup.-). These mutants contained a single C to T transition in the nodD coding sequence, substituting an asparagine for an aspartic acid in the nodD protein. Other nodD mutant classes affected either in autoregulation or the ability to activate nod gene induction, or both, were also described. It is suggested that the overproduction of the inducible nod gene in class IV mutants is deleterious to nodulation, resulting in decreased nodule number and ability to fix nitrogen. The Rhizobium leguminosarum nodD mutants described by Burn et al. (1987) supra were isolated by induced mutagenesis of plasmids containing nodD. The mutagenized nodD plasmids were then introduced into R. leguminosarum strains which lacked a Sym-plasmid and contained either a nodC- or nodD-lacZ fusion. Mutants affected in induction response were screened employing expression of .beta.-galactosidase to assess nod gene induction.